Binesh Unnikrishnan

A simple electrochemical approach to fabricate a glucose biosensor based on graphene–glucose oxidase biocomposite

We report a simple electrochemical approach for the immobilization of glucose oxidase (GOx) on reduced graphene oxide (RGO). The immobilization of GOx was achieved in a single step without any cross linking agents or modifiers. A simple solution phase approach was used to prepare exfoliated graphene oxide (GO), followed by electrochemical reduction to get RGO-GOx biocomposite. The direct electrochemistry of GOx was revealed at the RGO-GOx modified glassy carbon electrode (GCE). The electrocatalytic and electroanalytical applications of the proposed film were studied by cyclic voltammetry (CV) and amperometry. It is notable that the glucose determination has been achieved in mediator-free conditions. RGO-GOx film showed very good stability, reproducibility and high selectivity. The developed biosensor exhibits excellent catalytic activity towards glucose over a wide linear range of 0.1-27mM with a sensitivity of 1.85μAmM(-1)cm(-2). The facile and easy electrochemical approach used for the preparation of RGO-GOx may open up new horizons in the production of cost-effective biosensors and biofuel cells.

One-step synthesis of biofunctional carbon quantum dots for bacterial labeling

In this study, we used a simple one-step dry heating method to synthesize mannose-modified fluorescent carbon quantum dots (Man-CQDs) from solid ammonium citrate and mannose, and successfully applied for labeling Escherichia coli. The highly soluble Man-CQDs had an average particle diameter of 3.1±1.2 nm and exhibited a quantum yield of 9.8% at excitation and emission wavelengths of 365 and 450 nm, respectively. The fluorescent Man-CQDs could selectively bind to the FimH lectin unit in the flagella of the wild-type 1 E. coli K12 strain. We optimized the labeling efficiency of the Man-CQDs by controlling the ratio of ammonium citrate to mannose during their synthesis. The specific binding of the mannose units to E. coli allowed quantitative detection of the bacteria at levels down to 450 colony forming units mL(-1) in lab samples, and facilitate the application of the Man-CQDs for bacterial analyses of real samples (tap water, apple juice, human urine). The synthesis of our Man-CQDs, their labeling, and their use in the detection of bacteria were all simple, inexpensive and efficient processes.

Gold-nanoparticles-modified cellulose membrane coupled with laser desorption/ionization mass spectrometry for detection of iodide in urine.

We report an efficient method for the determination of iodide (I(-)) ions by using gold-iodide hybrid cluster ions on gold nanoparticles (Au NPs) modified mixed cellulose ester membrane (Au NPs-MCEM) by pulsed laser desorption/ionization mass spectrometry (LDI-MS). When I(-) ions were deposited and concentrated on the surfaces of Au NPs (32 nm) via strong Au(+)-I(-) interaction on the MECM, the Au NPs-MCEM was observed to function as an efficient surface-assisted LDI substrate with very low background noise. When pulsed laser radiation (355 nm) was applied, I(-) binding to Au NPs ions induced the enhancement of the desorption and ionization efficiency of gold-iodide hybrid cluster ions from the Au NPs surfaces. The reproducibility of the probe for both shot-to-shot and sample-to-sample (both less than 10%) ion production was also improved by the homogeneous nature of the substrate surface. Thus, it allows the accurate and precise quantification of I(-) ions in high-salinity real samples (i.e., edible salt samples and urine) at the nanomolar range. This novel LDI-MS approach provides a simple route for the high-speed analysis of I(-) ions with high sensitivity and selectivity in real biological samples.

Membrane-based assay for iodide ions based on anti-leaching of gold nanoparticles.

We report a label-free colorimetric strategy for the highly selective and sensitive detection of iodide (I(-)) ions in human urine sample, seawater and edible salt. A poly(N-vinyl-2-pyrrolidone)-stabilized Au nanoparticle (34.2-nm) was prepared to detect I(-) ions using silver (Ag(+)) and cyanide (CN(-)) ions as leaching agents in a glycine-NaOH (pH 9.0) solution. For the visual detection of the I(-) ions by naked eye, and for long time stability of the probe, Au nanoparticles (NPs) decorated mixed cellulose ester membrane (MCEM) was prepared (Au NPs/MCEM). The Au NPs-based probe (CN(-)/Ag(+)-Au NPs/MCEM) operates on the principle that Ag(+) ions form a monolyar silver atoms/ions by aurophilic/argentophilic interactions on the Au NPs and it accelerates the leaching rate of Au atoms in presence of CN(-) ions. However, when I(-) is introduced into this system, it inhibits the leaching of Au atoms because of the strong interactions between Ag/Au ions and I(-) ions. Inductively coupled plasma mass spectrometry, surface-assisted laser desorption/ionization time-of-flight mass spectrometry were used to characterize the surface properties of the Au NPs in the presence of Ag(+) and I(-). Under optimal solution conditions, the CN(-)/Ag(+)-Au NPs/MCEM probe enabled the detection of I(-) by the naked eye at nanomolar concentrations with high selectivity (at least 1000-fold over other anions). In addition, this cost-effective probe allowed the determination of I(-) ions in complex samples, such as urine, seawater, and edible salt samples.

Controlled synthesis, characterization and photocatalytic activity of BiPO4 nanostructures with different morphologies

The synthesis of bismuth phosphate (BiPO4) nanostructures with various morphologies and phases was explored under ultrasound irradiation and hydrothermal process. Powder x-ray diffraction (XRD), Raman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and UV–Vis diffuse reflectance spectroscopy (DRS) were used to characterize the BiPO4 samples. The effects of ultrasound irradiation and hydrothermal conditions on the phases and morphologies of the BiPO4 samples were studied, and the growth mechanisms of the 1D structure were investigated. The different BiPO4 samples exhibited different optical properties and photocatalytic activities for the degradation of methyl blue (MB) under UV light irradiation. The experimental results suggest that the high photocatalytic activity of the sample prepared under hydrothermal conditions is due to a low electron and hole recombination rate and the high potential of the photogenerated holes in the valence band. The practicality of this BiPO4 photocatalyst was validated for the degradation of MB in environmental and industrial wastewater samples, which demonstrated the advantages of its high photocatalytic activity.

Synthesis of Self‐Assembled Spermidine‐Carbon Quantum Dots Effective against Multidrug‐Resistant Bacteria

This study reports a two-step method to synthesize spermidine-capped fluorescent carbon quantum dots (Spd-CQDs) and their potential application as an antibacterial agent. Fluorescent carbon quantum dots (CQDs) are synthesized by pyrolysis of ammonium citrate in the solid state and then modified with spermidine by a simple heating treatment without a coupling agent. Spermidine, a naturally occurring polyamine, binds with DNA, lipids, and proteins involved in many important processes within organisms such as DNA stability, and cell growth, proliferation, and death. The antimicrobial activity of the as-synthesized Spd-CQDs (size ≈4.6 nm) has been tested against non-multidrug-resistant E. coli, S. aureus, B. subtilis, and P. aeruginosa bacteria and also multidrug-resistant bacteria, methicillin-resistant S. aureus (MRSA). The minimal inhibitory concentration value of Spd-CQDs is much lower (>25 000-fold) than that of spermidine, indicating their promising antibacterial characteristics. The mechanism of antibacterial activity is investigated, and the results indicate that Spd-CQDs cause significant damage to the bacterial membrane. In vitro cytotoxicity and hemolysis analyses reveal the high biocompatibility of Spd-CQDs. To demonstrate its practical application, in vitro MRSA-infected wound healing studies in rats have been conducted, which show faster healing, better epithelialization, and formation of collagen fibers when Spd-CQDs are used as a dressing material.

Functional gold nanoparticles coupled with laser desorption ionization mass spectrometry for bioanalysis

Laser desorption/ionization (LDI) coupled with mass spectrometry (MS) has evolved as an important tool for the analysis of a wide range of analytes, from small molecules to biomacromolecules. The LDI-MS technique requires a suitable matrix or substrate to absorb laser energy for the desorption and ionization of analytes. LDI-MS performed with an organic matrix easily suffers from many matrix effects, such as the formation of many adduct ions, high noise in the low-molecular-weight region, and poor reproducibility. Nanomaterials can reduce the interference of matrix molecules, and improve the reproducibility, sensitivity and even selectivity of LDI-MS. Among the nanomaterial substrates, gold nanoparticles (Au NPs) are an excellent nano-substrate for LDI-MS due to their high photoabsorption, photothermal conversion, and heat-transfer efficiency. Moreover, substantial amount of surface ligand ions and Au cluster ions are formed during the LDI process. By monitoring the ligand ions and Au cluster ions, it is possible to amplify the mass spectral signals and analyze macromolecules with minimum errors. This review highlights some of the applications of functionalized Au NPs coupled with LDI-MS for signal amplification in biosensing and bioimaging applications.

Monitoring Thrombin Generation and Screening Anticoagulants through Pulse Laser-Induced Fragmentation of Biofunctional Nanogold on Cellulose Membranes

Thrombin generation (TG) has an important part in the blood coagulation system, and monitoring TG is useful for diagnosing various health issues related to hypo-coagulability and hyper-coagulability. In this study, we constructed probes by using mixed cellulose ester membranes (MCEMs) modified with gold nanoparticles (Au NPs) for monitoring thrombin activity using laser desorption/ionization mass spectrometry (LDI-MS). The LDI process produced Au cationic clusters ([Au(n)](+); n = 1-3) that we detected through MS. When thrombin reacted with fibrinogen on the Au NPs-MCEMs, insoluble fibrin was formed, hindering the formation of Au cationic clusters and, thereby, decreasing the intensity of their signals in the mass spectrum. Accordingly, we incorporated fibrinogen onto the Au NPs-MCEMs to form Fib-Au NPs-MCEM probes to monitor TG with good selectivity (>1000-fold toward thrombin with respect to other proteins or enzymes) and sensitivity (limit of detection for thrombin of ca. 2.5 pM in human plasma samples). Our probe exhibited remarkable performance in monitoring the inhibition of thrombin activity by direct thrombin inhibitors. Analyses of real samples using our new membrane-based probe suggested that it will be highly useful in practical applications for the effective management of hemostatic complications.

Visual detection of cyanide ions by membrane-based nanozyme assay

In this paper, we report a simple one-step synthesis of well-dispersed amorphous cobalt hydroxide/oxide-modified graphene oxide (CoOxH-GO) possessing peroxidase-like catalytic activity, and its application for the detection of H2O2, glucose, and CN- ions. CoOxH is formed and deposited in situ on the GO surface through the reaction between GO (size ~ 240nm) and Co2+ in basic solution at room temperature. We investigated the enzyme-mimicking activity of the CoOxH-GO nanohybrid in detail via the H2O2-mediated oxidation of Amplex Red (AR) to form fluorescent resorufin. The peroxidase-like activity of CoOxH-GO is utilized herein for the quantitation of H2O2 in a wide concentration range, from 100nM to 100μM. When coupled with glucose oxidase (GOD), the AR/CoOxH-GO system can determine glucose level in blood samples. Interestingly, cyanide ions (CN-) significantly inhibit the catalytic activity of the CoOxH-GO nanohybrid, which allows for the construction of a probe for the detection of CN- in water samples and laboratory wastes. We fabricated a membrane-based CoOxH-GO probe for the visual detection of CN- by preparing a thin film of CoOxH-GO on a positively charged and porous nylon membrane (N+M). The CoOxH-GO/N+M operates on the principle that CN- inhibits the catalytic activity of CoOxH-GO towards the H2O2-mediated oxidation of AR to form reddish resorufin on the membrane. The intensity of the red color of the membrane decreases with increasing CN- concentration, which can be easily observed with the naked eye at the nanomolar level. This cost-effective sensing system allows for the rapid and simple determination of the concentrations of CN- in complicated wastewater samples.

Detection of urinary spermine by using silver-gold/silver chloride nanozymes

In this paper, we report a simple one-step synthesis method for silver-gold bimetallic nanoparticles deposition on silver chloride nanosheets to form Ag-Au/AgCl nanohybrid with oxidase-like and peroxidase-like catalytic activity. We used these nanohybrid in the detection of spermine. First, 13 nm-sized Au NPs were synthesized by citrate reduction of HAuCl4 solution, and then, Ag+ ions were added to the solution without any purification. The added Ag+ reacted with the Cl- ions in the dispersion, thus immediately forming AgCl nanosheets through a precipitation reaction, and the aurophilic interactions with the Au NPs resulted in the formation and in situ self-deposition of Ag-Au NPs on the AgCl nanosheets at room temperature. We investigated the enzyme-mimicking activity of the Ag-Au/AgCl nanohybrid in detail via the O2- or H2O2-Amplex Red (AR) redox system. The Ag-Au/AgCl nanohybrid exhibited at least 150-fold higher catalytic activity than that of Ag-Au NPs or AgCl nanosheets, due to synergistic effect. Spermine inhibited the enzyme-mimic activity of the Ag-Au/AgCl nanohybrid, thereby allowing for the construction of a probe for detecting nanomolar concentrations of spermine in urine samples. This cost-effective sensing system was used to easily and rapidly detect the concentrations of spermine in complex urine samples.